The technique of fluorescence photobleaching recovery is used to determine the diffusion coefficients of fluorescently labeled molecules. A three-dimensionally defined spot of dye is bleached out and the fluorescence recovery is monitored as unbleached molecules diffuse back into the spot. The shape of the recovery curve yields the diffusion coefficient and information about immobile fraction and particle flow. Conventional one-photon fpr is restricted to the study of cell membranes or very thin regions of cells (lamellapodia) because the laser beam is able to excite fluorescence all along the beam length. In order to excite a three-dimensionally defined volume the third dimension must be limited by the sample itself. Two photon fluorescent excitation is confined to a three-dimensionally defined ellipsoid around the focal spot. This enables TPFPR to be performed within the cytoplasm of living cells. We have developed a fast user-friendly instrument that measures the diffusion coefficient of fluorescently labeled molecules in solution, and have verified its performance by measuring the diffusion coefficient of fluorescein-labeled BSA, finding a value of 6.8 e-7 cm^2 /s, in excellent agreement with published values.